PROJECT SUMMARY Protein synthesis in mammalian cells depends not only on the transcription of RNA from the DNA genome, but also on the stability of that RNA and its rate of translation. A class of ~21 nucleotide RNAs known as ?microRNAs? play critical roles in regulating protein synthesis by recognizing complementary sequences in protein-coding RNA molecules and causing either RNA cleavage or inhibition of translation. MicroRNA regulation plays key roles in nearly every studied human physiological system, and mis- regulation of individual microRNAs or global microRNA processing has been linked to cancer, cardiac and kidney disease, viral infection response, and many other diseases. Identifying the targets of individual microRNAs provides an essential insight into the functional biological role of each microRNA. Furthermore, as functional regulatory molecules microRNAs are now being highly pursued for their use as therapeutic agents, where the ability to directly map microRNA targets is required to assay both proper on-target binding as well as low off-target interactions. However, current methods are lacking, as they have either high false-positive rates, lack the ability to assign targets to individual microRNAs, or do not scale to profiling all (particularly low-abundance) microRNAs with quantitative accuracy. Recently we developed the enhanced CLIP-seq (eCLIP) methodology, with 1000-fold improved efficiency of generating high-throughput sequencing libraries from RBP profiling experiments, enabling highly robust and reproducible RBP target profiling through the incorporation of paired size-matched inputs. Eclipse has successfully developed eCLIP as a highly profitable contract service product, and now has an eCLIP kit in beta testing. Here we will develop a specialized variant of the eCLIP method for unambiguous mapping of microRNA targets transcriptome-wide in the following three aims: 1. Validate unambiguous direct profiling of miRNA targets with chimeric eCLIP (chim-eCLIP). 2. Validate simplified chim-eCLIP method for commercialization. 3. Conversion of chimeric eCLIP into a structured and well-documented kit format. Eclipse Bio is an ideal candidate to perform the aims described above due to our expertise in genomics and computational biology, particularly in RNA processing and profiling RNA binding protein targets. The three aims above will enable microRNA target mapping to be performed in a standard method by all biomedical researchers in academia and industry, and create a rigorous standard for validating specificity of therapeutic microRNAs. The ability to properly assess therapeutic miRNA-like molecules will provide significant benefits to researchers studying microRNA regulation in various biological contexts and drug companies developing RNA therapies in the clinic. Additionally, in San Diego we are close to many research institutes and biotechs doing RNA research that can provide scientific and commercialization expertise and assistance.